Biological indicators (BIs) are used to monitor the efficacy of sterilization processes for medical products. BIs contain high numbers of bacterial spores (generally 104 to 106 that are highly resistant to the sterilization process for which they are designed. This paper is Part II of a series reporting on the range and distribution of grow-out times for BIs exposed to sublethal sterilization processes. The authors describe studies to confirm and extend their original findings that the grow-out times for a set of nonsterile BIs approximates a normal distribution and that one or more BIs from such a set would occasionally exhibit delayed outgrowth.

This paper is Part II of a series reporting on the range and distribution of grow-out times for biological indicators (BIs) exposed to sublethal sterilization processes. Part I described grow-out times for self-contained BIs with Geobacillus stearothermophilus spores on paper carriers that were exposed to moist-heat sterilization processes at 121 °C (1). The studies described in this paper were performed to confirm and extend the authors' original findings that the grow-out times for a set of nonsterile BIs approximates a normal distribution, and occasionally, one or more BIs from such a set would exhibit a delayed outgrowth. Delayed nonsterile BIs were almost exclusively found in sets of BIs where the average number of surviving colony-forming units (CFU) was estimated at less than 1.0/BI unit.

The previous studies exposed BIs to moist-heat sterilization processes that gave results that met the FDA protocol for establishing a reduced incubation time (RIT) (2). Sets of exposed BIs that have 30 to 80 nonsterile out of the 100 tested have a range estimate of 0 to 5 CFU per BI (1). In the studies reported in this article, the FDA RIT protocol was again followed, but testing was also performed using exposures based on the calculated survival time that is referenced in the US Pharmacopeia (USP) and Association for the Advancement of Medical Instrumentation (AAMI)/International Organization for Standardization (ISO) documents (3–5). The calculated survival exposure time results in BIs with approximately 100 surviving CFU, a much higher average value when compared to the results found after exposure of BIs to the conditions required to meet the FDA RIT protocol.

For the studies reported in this article, the moist-heat sterilization processes tested were expanded to include exposures at 132 °C, 134 °C, and 135 °C. BIs exposed to hydrogen peroxide (H2O2) vapor were self-contained with 106 spores of G. stearothermophilus inoculated on stainless-steel discs. BIs exposed to ethylene oxide (EO) gas and chlorine dioxide (ClO2) gas sterilization processes were also tested. For the EO and CIO2 processes, BIs with Bacillus atrophaeus spores were used; both paper spore strips and self-contained BIs with paper carriers were tested.